Cyclic di-GMP (hereinafter, sometimes called “c-di-GMP”) is a bacterial signal molecule involved in biofilm formation, motility, virulence factor expression, and the like of bacteria, and the physiological activity, the signaling mechanism, and the like thereof receive attention in recent years. For example, c-di-GMP was found to have immunostimulatory action and is used as an adjuvant, an active ingredient of an allergy-regulating drug, and the like, thereby being recently expected to be promising as a pharmaceutical.
C-di-GMP has been only limitedly available and very expensive. Because of this, in order to produce c-di-GMP efficiently, chemical synthesis and enzymatic synthesis have been conventionally studied and, among these, enzymatic synthesis has been considered to be practical as the production method.
C-di-GMP can be synthesized from two GTP molecules by a two-step enzymatic reaction via the catalytic action of a diguanylate cyclase (hereinafter, sometimes called “DGC”). DGCs derived from various living organisms have “GGDEF (SEQ ID NO:26) domain”, which is responsible for the activity and is well conserved among biological species. GGDEF (SEQ ID NO:26) domain has a region called an i-site that is known to be involved in product inhibition in a c-di-GMP synthesis reaction. Various living organisms have a gene that has GGDEF (SEQ ID NO:26) domain and is supposed to have DGC activity. The GGDEF (SEQ ID NO:26) domains have high homology with each other, while the regions other than the GGDEF (SEQ ID NO:26) domains vary to a great extent among the genes and assume different structures.
As a known, conventional method for enzymatically synthesizing c-di-GMP, a one-pot synthesis of c-di-GMP is disclosed where a mutant DGC that is derived from Caulobacter crescentus and results from expression in Escherichia coli or the like in a large amount in the form of an inclusion body is used along with a guanylate kinase and a nucleoside diphosphate kinase (Patent Document 1). With its c-di-GMP production process requiring a step of purifying an inclusion body from an Escherichia coli cell and a step of refolding the resultant DGC in the form of an inclusion body for reactivation, the method in Patent Document 1 has too many complicated treatment processes for large-scale industrial synthesis, thereby having many problems.
As another known method, use of a DGC derived from thermophilic bacterium Thermotoga maritima is known (Patent Document 2 and Non-patent Document 1). It is described that synthesis in this method used a modified enzyme obtained by exclusively expressing GGDEF (SEQ ID NO:26) domain, which is the minimum functional region, of a DGC derived from T. maritima and, into the i-site, introducing amino acid mutation so as to remove product inhibition.
The inventors of the present invention reported development of a c-di-GMP synthesis system where a DGC derived from Geobacillus stearothermophilus (hereinafter, sometimes called “GsDGC”) is used (Non-patent Document 2). Non-patent Document 2 describes a combined use of polyphosphate:AMP phosphotransferase derived from Acinetobacter johnsonii and a polyphosphate-dependent nucleoside diphosphate kinase derived from Pseudomonas aeruginosa in the enzymatic reaction system to develop a system for supplying GTP from GMP provided by polyphosphoric acid serving as a donor. The synthesis system in Non-patent Document 2 does not need frequent GTP addition that is necessary in the synthesis system in Patent Document 2 and Non-patent Document 1 due to substrate inhibition caused by GTP, and is therefore an excellent synthesis system. As GTP is expensive, the synthesis system in Non-patent Document 2 has cost advantage as well.